Project Summary/Abstract Piwi proteins and Piwi-interacting RNAs (piRNAs) are essential for animal fertility because they repress transposable elements (TE) and regulate germ cell-specific genes. Although vertebrate genomes encode hundreds of piRNA cluster loci, we know little about the impact and gene regulation roles of individual piRNA clusters. The reason is because Piwi pathway mutants disrupt all piRNAs and cause widespread germ cell death, thereby obscuring the dissection of functions for individual piRNA Cluster loci. Major knowledge gaps include: (1) Which genes besides TEs impact fertility and are being targeted by PIWI/piRNA complexes? (2) Will a single piRNA cluster mutation affect the production of piRNAs from other clusters? (3) How will the regulation of Piwi targets and vertebrate germ cell development be affected by mutating a single piRNA cluster? This exploratory project will address these questions by first generating a list of Xenopus Piwi- associated transcripts (PATs) and testing their mechanism of regulation in Xenopus oocyte extracts. We will then create an intergenic piRNA cluster locus mutant in Xenopus tropicalis, and examine which PATs are affected in this mutant so as to gain insight into the relationship between specific groups of piRNAs and target genes regulated by Piwi/piRNA complexes. The frog is an experimentally tractable vertebrate system for dissecting the role of individual piRNA clusters. These Xenopus studies will reveal how a single major intergenic piRNA cluster will affect gonadogenesis and embryonic development. The future characterizations of mutant phenotypes are considerations for a longer-term project after the initiation of this exploratory project. The focus of this exploratory project is to create a list of PATs, test for PAT regulation in oocyte extracts, and then create a valuable mutant that will enable precise examination of which PATs are mis-regulated. Given our foundation of expertise described in this proposal, we are confident in achieving the goals of this exploratory project.